Lactobacillus paracasei strain and use thereof

ABSTRACT

Human-derived probiotic strains  Lactobacillus paracasei  KBL382,  Lactobacillus paracasei  KBL384 and  Lactobacillus paracasei  KBL385 are described, as well as uses of the strains for improving intestinal health and treating or preventing intestinal diseases. The strains have excellent anti-inflammatory and immunomodulatory functions, superb strengthening effects on tight junctions of the intestinal tract wall, suppress enteritis-induced weight loss and colon length reduction, thereby exhibiting therapeutic effects for enteritis, and significantly alleviate the symptoms of atopic dermatitis. The strains can be used as probiotic material for enhancing anti-inflammatory effects, strengthening immunity, improving intestinal health functions, and alleviating allergic diseases.

RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/KR2019/005553, which designates the United States and was filed onMay 9, 2019, published in Korean and claims priority under 35 U.S.C. §119 or 365 to Korean Application No. 10-2018-0053279, filed May 9, 2018.The entire teachings of the above applications are incorporated hereinby reference.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

This application incorporates by reference the Sequence Listingcontained in the following ASCII text file:

a) File name: 58881000001 SEQUENCE LISTING; created Nov. 6, 2020, 9 KBin size.

TECHNICAL FIELD

The present invention relates to strains of Lactobacillus paracasei andthe use thereof. More specifically, the present invention relates to ahealth functional food composition for alleviation of allergic symptoms,improvement of immunoregulation, alleviation of inflammatory symptoms,alleviation of atopic dermatitis, and improvement of intestinal health;and a pharmaceutical composition for the treatment or prevention ofallergic diseases, autoimmune diseases, inflammatory diseases, atopicdermatitis and/or intestinal diseases, comprising an effective amount ofat least one selected from the group consisting of a Lactobacillusparacasei KBL382 strain, a Lactobacillus paracasei KBL384 strain, aLactobacillus paracasei KBL385 strain, microbial cells of said strain,cultures of said strain, lysates of said strain, and extracts of saidstrain.

BACKGROUND

Probiotics refer to microorganisms and the resulting products therefromhaving anti-bacterial activities and enzyme activities to help thebalance of intestinal microorganisms. In addition, probiotics are alsodefined as live bacteria in the form of a single or multiple strain(s)to improve intestinal flora when provided to human or animals in theform of dry cells or fermentation products. Probiotics must inhabit thehuman gut, be non-pathogenic and non-toxic, and survive long enoughuntil they arrive at the intestine. Further, probiotics must maintainviability and activities until they are consumed in the food delivered,be sensitive to antibiotics used to prevent infection, and not haveantibiotic-resistant plasmids. Also, probiotics must be resistant toacids, enzymes, and bile in the intestinal environment.

These probiotics may include, for example, Bacillus sp. having anexcellent ability to produce digestive enzymes such as amylase,protease, lipase, cellulase, and phosphatase, Lactobacillus sp.producing lactic acid, and photosynthetic bacteria preventing stink byway of using the stink-causing substances (such as ammonia, hydrogensulfide, and amines) remaining in the feces of livestock in metabolicprocess.

In particular, Bacillus sp. and Lactobacillus sp. are known as veryuseful probiotics because they include strains that produce variousantibacterial substances. These lactic acid bacteria produceantimicrobial peptides called Bacteriocin, which have antibacterialmechanisms that are not related to the mechanism of antibioticresistance. Bacteriocins have polymorphic characteristics that theirmolecular weights, biochemical properties, and antimicrobial ranges andmechanisms for the host vary considerably. Klaenhammer definesbacteriocin as a protein or protein complex that has directantibacterial activity against species close to bacteriocin-producingbacteria.

Meanwhile, irritable bowel syndrome (IBS) is a symptom characterized byabdominal pain, and/or irritations associated with changed intestinalmovement or bowel habits, such symptoms cannot be explained withanatomic or biochemical abnormality. Common symptoms of IBS also includeurinary urgency, bloating and feeling of incomplete intestinal movement.Accordingly, IBS can be classified as functional gastrointestinaldisorders comprising conditions such as functional bloating, non-cardiacchest pain, non-ulcerative dyspepsia, and chronic constipation ordiarrhea. In particular, in the case of IBS, since the related symptomsaffect both well-being and normal functional aspect of patients, thedisease has a huge impact on morbidity and quality of life, beyondabdominal pain and discomfort.

Inflammatory bowel disease (IBD) is a condition in which abnormalchronic inflammation in the intestine repeats improvement andrecurrence, comprising all intestinal inflammatory diseases, such asCrohn's disease, ulcerative colitis, or Behcet's disease, but notlimited thereto. Many researches have been conducted in the field ofdrug development to treat IBS and IBD. In this regard, variousantidepressants are commonly used, even though the efficacy thereof inclinical trials is moderate and the clinical utility thereof is limiteddue to significant side effects. Serotonergic medications have also beenproved to have efficacy against overall IBS symptoms. However, theapplication of these medications has been restricted in various ways dueto recent several safety problems. Accordingly, there is increasinginterest in developing a new therapeutic agent for IBS.

Meanwhile, allergy is a biochemical phenomenon that exhibits a unique,altered response to a foreign substance (antigen, allergen). The foreignsubstance which causes symptoms is called allergen, while the diseasesfrom those symptoms are called allergic diseases. Allergy is apathological process in the living body resulting from theantigen-antibody reaction. In general, there are four types of allergiesdepending on the period to trigger the reaction and the complementinvolvement. Type 1, among those, is anaphylactic type (immediate type)in which target organs are mostly digestive organs, skin and lungs, andthe common symptoms include gastrointestinal allergy, urticaria,atrophodermatitis, allergic rhinitis, and bronchial asthma, etc. Thepathological mechanism of Type 1 is known as follows: when antigenscontact IgE antibodies attached to the surface of mast cells andbasophilic leukocytes, the target cells are activated to secretechemical transmitters such as histamine, leukotriene, and PAF, and thenblood vessels and smooth muscles are contracted. Such mechanism can beoften combined with Type 4 (delayed type). In other words, suchanaphylaxis and allergic reaction can arise due to a variety of changesin the mast cells, etc. The activation of mast cells, which leads todegranulation, is caused by binding of antigen, anti-IgE, lectin, etc.to Fc receptors, stimulation of anaphylatoxin, etc., or other drugs suchas calcium ionophore, compound 48/80, codeine and syntheticadrenocorticotropic hormone.

Mast cells and basophils in blood are known as main cells in the body tocause many allergic diseases such as allergic rhinitis, allergicdermatitis, asthma, food allergy and anaphylactic shock. These cellshave receptors (FcRI) for IgE on their surfaces which is an antibodycausing allergy, and the cells are stimulated by the allergy-causingsubstances (antigen, allergen) to secrete their own variousallergy-causing substances out of the cells (Kim K et al, Eur JPharmacol, 581:191-203, 2008).

Among allergic diseases, atopic dermatitis, as widely known to thepublic, is a chronic recurrent skin disease that affects newborns orchildren and may persist until adulthood. Like asthma or allergicrhinitis, atopic dermatitis is an inflammatory skin disease associatedwith local infiltration of T-lymphocyte which produces IL-4 and IL-5.IL-4 controls the development of the T helper 2 (Th2) phenotype,resulting in overproduction of immunoglobulins (Ig) and eosinophilia,and increase of serum IgE levels. 80-90% of the subjects who werepositive to the skin test regarding food and inhalant allergens werefound to have atopic dermatitis.

There are different treatments for treating or preventing allergicdiseases including atopic dermatitis, but no effective treatment hasbeen found yet. Some drug-based treatments are known, but even a shortterm administration of the drug for the treatment would develop atolerance and a long-term administration may cause serious side effects,and thus such drug-based treatments of allergic diseases have beenavoided recently. Under the circumstances, without treatment having anyabsolute, obvious effect, irritating symptoms such as itching andredness of skin in addition to allergy often fail to improve.

WO 96/29083 and EP 554418 disclose two types of Lactobacillus strainswhich form colonies in bowel, i.e., Lactobacillus plantarum 299v (DSM6595) and Lactobacillus casei ssp. rhamnosus 271 (DSM 6594), etc. EP415941 discloses a method for preparing a nutrient composition,comprising treating oat gruel with enzymes before mixing it withlactobacilli. U.S. Pat. No. 7,195,906 discloses a strain ofBifidobacterium isolated from resected and washed human gastrointestinaltract for the treatment of inflammatory diseases, especiallygastrointestinal inflammation such as IBD and IBS.

However, no strain having excellent effects on improving intestinalhealth, for example, treatment of IBD and IBS, and on alleviatingallergic symptoms has been found yet, and in order to find strainshaving such effects, many research institutions have been working on.

Under the circumstances, the present inventors screened a variety ofstrains based on the fact that a health improvement effect of probioticsis not general attributes of genus and species but rather is strainspecific (Report of a joint FAO/WHO working group on drafting guidelinesfor the evaluation of probiotics in food, London Ontario, Canada, 2002),and identified a novel strain having excellent effects onimmunoregulation, anti-inflammation activities, alleviation of allergy,improvement of intestinal health and treatment of intestinal diseases.Then, the present invention was completed by confirming superior effectsof these strains.

SUMMARY

The purpose of the present invention is to provide a novel strain usefulfor improvement of intestinal health, treatment or prevention ofintestinal diseases, improvement of immunoregulation, treatment orprevention of autoimmune diseases, alleviation of allergic symptoms,alleviation of inflammatory symptoms, alleviation and treatment ofatopic dermatitis.

In order to achieve the purpose, the present invention provides aLactobacillus paracasei KBL382 strain (Accession No. KCTC13509BP), aLactobacillus paracasei KBL384 strain (Accession No. KCTC13510BP), or aLactobacillus paracasei KBL385 strain (Accession No. KCTC13511BP).

Also, the present invention provides a food composition or food additivecomposition comprising an effective amount of at least one selected fromthe group consisting of a Lactobacillus paracasei KBL382 strain(Accession No. KCTC13509BP), a Lactobacillus paracasei KBL384 strain(Accession No. KCTC13510BP), or a Lactobacillus paracasei KBL385 strain(Accession No. KCTC13511BP), cultures of said strain, lysates of saidstrain, and extracts of said strain.

The present invention also provides a pharmaceutical composition for thetreatment or prevention of intestinal diseases, comprising an effectiveamount of at least one selected from the group consisting of aLactobacillus paracasei KBL382 strain (Accession No. KCTC13509BP), aLactobacillus paracasei KBL384 strain (Accession No. KCTC13510BP), or aLactobacillus paracasei KBL385 strain (Accession No. KCTC13511BP),cultures of said strain, lysates of said strain, and extracts of saidstrain.

The present invention also provides a pharmaceutical composition for thetreatment or prevention of allergic diseases, comprising an effectiveamount of at least one selected from the group consisting of aLactobacillus paracasei KBL382 strain (Accession No. KCTC13509BP), aLactobacillus paracasei KBL384 strain (Accession No. KCTC13510BP), or aLactobacillus paracasei KBL385 strain (Accession No. KCTC13511BP),cultures of said strain, lysates of said strain, and extracts of saidstrain.

The present invention also provides a pharmaceutical composition for thetreatment or prevention of autoimmune diseases or inflammatory diseases,comprising an effective amount of at least one selected from the groupconsisting of a Lactobacillus paracasei KBL382 strain (Accession No.KCTC13509BP), a Lactobacillus paracasei KBL384 strain (Accession No.KCTC13510BP), a Lactobacillus paracasei KBL385 strain (Accession No.KCTC13511BP), cultures of said strain, lysates of said strain, andextracts of said strain.

The present invention also provides a method for treating intestinaldiseases, comprising administering a therapeutically effective amount ofat least one selected from the group consisting of a Lactobacillusparacasei KBL382 strain (Accession No. KCTC13509BP), a Lactobacillusparacasei KBL384 strain (Accession No. KCTC13510BP), or a Lactobacillusparacasei KBL385 strain (Accession No. KCTC13511BP), cultures of saidstrain, lysates of said strain, and extracts of said strain to a subjectin need thereof.

The present invention also provides a method for treating allergicdiseases, comprising administering a therapeutically effective amount ofat least one selected from the group consisting of a Lactobacillusparacasei KBL382 strain (Accession No. KCTC13509BP), a Lactobacillusparacasei KBL384 strain (Accession No. KCTC13510BP), or a Lactobacillusparacasei KBL385 strain (Accession No. KCTC13511BP), cultures of saidstrain, lysates of said strain, and extracts of said strain to a subjectin need thereof.

The present invention also provides a method for treating autoimmunediseases or inflammatory diseases, comprising administering atherapeutically effective amount of at least one selected from the groupconsisting of a Lactobacillus paracasei KBL382 strain (Accession No.KCTC13509BP), a Lactobacillus paracasei KBL384 strain (Accession No.KCTC13510BP), a Lactobacillus paracasei KBL385 strain (Accession No.KCTC13511BP), cultures of said strain, lysates of said strain, andextracts of said strain to a subject in need thereof.

The present invention also provides a composition for the use ofpreventing or treating intestinal diseases, comprising at least oneselected from the group consisting of a Lactobacillus paracasei KBL382strain (Accession No. KCTC13509BP), a Lactobacillus paracasei KBL384strain (Accession No. KCTC13510BP), or a Lactobacillus paracasei KBL385strain (Accession No. KCTC13511BP), cultures of said strain, lysates ofsaid strain, and extracts of said strain.

The present invention also provides a composition for the use ofpreventing or treating allergic diseases, comprising at least oneselected from the group consisting of a Lactobacillus paracasei KBL382strain (Accession No. KCTC13509BP), a Lactobacillus paracasei KBL384strain (Accession No. KCTC13510BP), or a Lactobacillus paracasei KBL385strain (Accession No. KCTC13511BP), cultures of said strain, lysates ofsaid strain, and extracts of said strain.

The present invention also provides a composition for the use ofpreventing or treating autoimmune diseases or inflammatory diseases,comprising at least one selected from the group consisting of aLactobacillus paracasei KBL382 strain (Accession No. KCTC13509BP), aLactobacillus paracasei KBL384 strain (Accession No. KCTC13510BP), or aLactobacillus paracasei KBL385 strain (Accession No. KCTC13511BP),cultures of said strain, lysates of said strain, and extracts of saidstrain.

The present invention also provides the use of a composition comprisingat least one selected from the group consisting of a Lactobacillusparacasei KBL382 strain (Accession No. KCTC13509BP), a Lactobacillusparacasei KBL384 strain (Accession No. KCTC13510BP), a Lactobacillusparacasei KBL385 strain (Accession No. KCTC13511BP), cultures of saidstrain, lysates of said strain, and extracts of said strain, forpreparing a preventive or therapeutic drug for intestinal diseases.

The present invention also provides the use of a composition comprisingat least one selected from the group consisting of a Lactobacillusparacasei KBL382 strain (Accession No. KCTC13509BP), a Lactobacillusparacasei KBL384 strain (Accession No. KCTC13510BP), or a Lactobacillusparacasei KBL385 strain (Accession No. KCTC13511BP), cultures of saidstrain, lysates of said strain, and extracts of said strain, forpreparing a preventive or therapeutic drug for allergic diseases.

The present invention also provides the use of a composition comprisingat least one selected from the group consisting of a Lactobacillusparacasei KBL382 strain (Accession No. KCTC13509BP), a Lactobacillusparacasei KBL384 strain (Accession No. KCTC13510BP), or a Lactobacillusparacasei KBL385 strain (Accession No. KCTC13511BP), cultures of saidstrain, lysates of said strain, and extracts of said strain, forpreparing a preventive or therapeutic drug for autoimmune diseases orinflammatory diseases.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1E illustrate the results confirming the effect of controllinginflammatory cytokines by a KBL382 strain, a KBL384 strain and a KBL385strain of the present invention in PBMC.

FIGS. 2A-2D illustrate the results confirming the effect of controllinggene expression of T cell differentiation markers by a KBL382 strain, aKBL384 strain and a KBL385 strain of the present invention in PBMC.

FIGS. 3A-3F illustrate the results of observing the change in expressionlevel of cytokines, (FIG. 3A) IL-2, (FIG. 3B) IFN-γ, (FIG. 3C) IL-4,(FIG. 3D) IL-13, (FIG. 3E) IL-17A and (FIG. 3F) IL-10, according to theadministration of a KBL382 strain, a KBL384 strain and a KBL385 strainof the present invention, in PBMC cell lines that T-cells are activatedby addition of anti CD3 antibody.

FIGS. 4A-4E illustrate the result confirming effects on alleviatingenteritis through (FIG. 4A) body weight change, (FIG. 4B) the colonlength change, (FIG. 4C) the colon length change and cecum weightchange, (FIG. 4D) H&E staining of colon tissues and (FIG. 4E) the changein the MPO (myeloperoxidase) level in the colon tissues afteradministering each of a KBL382 strain, a KBL384 strain and a KBL385strain of the present invention to mouse models that enteritis wasinduced.

FIGS. 5A-5C illustrate the results of observing the effect ofstrengthening tight junctions of a KBL382 strain and a KBL385 strain ofthe present invention through the change in the expression level of theintestinal tract wall tight junction-related genes, (FIG. 5A) ZO1, (FIG.5B) Claudin3, and (FIG. 5C) MUC4.

FIGS. 6A-6C illustrate the results of comparing the effects of (FIGS. 6Aand 6B) recovering the length of colon and (FIG. 6C) improving bodyweight loss by a KBL382 strain of the present invention and infliximab,an antibody for treating enteritis, which is available in the market, inorder to confirm the effect of alleviating enteritis by the KBL382strain of the present invention.

FIGS. 7A-7F illustrate the results of observing (FIGS. 7A and 7B) thedermatitis score measurement, (FIG. 7C) the itching-alleviating effect,(FIGS. 7D and 7E) the skin thickness lowering effect and (FIG. 7F) theIgE concentration-in-blood lowering effect by the administration of aKBL382 strain of the present invention to animal models that atopicdermatitis was induced, in order to confirm the effect of alleviatingatopic symptoms by the KBL382 strain.

DETAILED DESCRIPTION

Unless defined otherwise, all of the technical, scientific terms used inthe present specification mean the same as understood by a person havingordinary skills in the art (“those skilled in the art”). In general, thenomenclature used in the present specification is well known in the artand commonly used.

The present invention has confirmed an immunoregulatory effect ofmicroorganisms derived from the human body, and found Lactobacillusparacasei strains having an excellent immunoregulation effect, i.e.,KBL382 (Accession No. KCTC13509BP), KBL384 (Accession No. KCTC13510BP)and KBL385 strain (Accession No. KCTC13511BP). Analysis of 16s rDNA ofsaid strain demonstrates that said strain is a novel strain which hasnever been known to the public.

In one embodiment, the present invention relates to a novel probioticstrain of Lactobacillus paracasei KBL382, of Lactobacillus paracaseiKBL384 or of Lactobacillus paracasei KBL385, and said strains arecharacterized by comprising 16s rDNA sequences of SEQ ID NOs: 1 to 3,respectively, as below.

16s rDNA sequence of a Lactobacillus paracasei KBL382 strain >KBL382<SEQ ID NO: 1> GCAGGTGGCGGGTGCTATACATGCAGTCGACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTGGGACTGTAGGCTGCAACTCGCCTAGACGAAGTCGGAATCGCTAGTAATCGGGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCGAAGCCGGTGGCGTAACCCTTTAGGGAGCGAGCGTCTAAGTGGCTCACGCCT 16s rDNA sequence of a Lactobacillus paracaseiKBL384 strain >KBL384 <SEQ ID NO: 2>GCCAGTGGGGGGGGTGCTATACATGCAGTCGAACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATGGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGGTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACAGGTGCTACAATGGATGGTACAAGGAGTTGCGAGACCGGGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCCCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCGAAGCCGGTGGCGTAACCCTTTAGGGAGCGAGCCGTCTAAGGTGAACCAAAGTTTG16s rDNA sequence of a Lactobacillus paracasei KBL385 strain >KBL385<SEQ ID NO: 3> GCAGTTGGGGGGGAGCTATACATGCAGTCGACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATGGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGCCGGGGTGAGSGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAAGTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTGGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGTTTCCCGTTGGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGCAGCGCAACCCTTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGGGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTGGCCTACAGGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTGCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCGAAGCCGGTGGCGTAACCCTTTAGGGAGCGAGCCGTCTAAGTGTACAAAGTT

The present invention also confirmed that a KBL382 strain, a KBL384strain, and a KBL385 strain each have a strengthening effect on tightjunction of the intestinal tract wall in addition to anti-inflammatoryand immunoregulatory effects, and that the administration of a KBL382strain, a KBL384 strain and a KBL385 strain to animal models thatenteritis was induced remarkably alleviated the body weight loss and thecolon length reduction due to enteritis. Further, it has also beenconfirmed that the administration of the KBL382 strain, among the abovestrains, to animal models that atopic dermatitis was induced remarkablyimproved the dermatitis score, alleviated itching symptoms, lowered theskin thickness and remarkably improved the IgE concentration-in-blooddue to allergic reaction.

Accordingly, in another embodiment, the present invention relates to afood composition or food additive composition comprising an effectiveamount of at least one selected from the group consisting of microbialcells of a KBL382 strain, a KBL384 strain, or a KBL385 strain, culturesof said strain, lysates of said strain, and extracts of said strain.

Said food composition or food additive composition can be readilyutilized as the food effective for improvement of intestinal health andprevention of intestinal diseases, for example, as main ingredients orminor ingredients of food, food additives, health functional foodcomposition or functional beverages, but not limited thereto.

Said food composition or food additive composition can be readilyutilized as the food effective for alleviation of allergic symptoms, forexample, as main ingredients or minor ingredients of food, foodadditives, health functional food composition or functional beverages,but not limited thereto.

Further, said food composition or food additive composition can bereadily utilized as the food effective for alleviation of autoimmunediseases or inflammatory diseases, for example, as main ingredients orminor ingredients of food, food additives, health functional foodcomposition or functional beverages, but not limited thereto.

The term “food” refers to a natural or artificial product comprising atleast one nutrient, and more preferably, refers to a product whichbecame edible through certain processing, usually encompassing all offood, food additives, health functional food and functional beverages.

The food that may comprise the said food composition according to thepresent invention as an additive may include, for example, differenttypes of food, beverages, chewing gum, tea, vitamin complex, andfunctional food. In addition, the food of the present invention includesspecial nutritional food (e.g., modified milk, infant/baby food),processed meat products, fish meat products, tofu, muk, noodles (e.g.,ramen, Asian noodles), bakery products, health supplement food,seasoning products (e.g., soy sauce, soybean paste, red pepper paste,mixed paste), sauces, confectionery (e.g., snack foods), candies,chocolates, chewing gums, ice-creams, milk products (e.g., fermentedmilk, cheese), other processed food, Kim-chi, salted food (e.g.,different types of Kim-chi, pickled food), beverages (e.g., fruit juice,vegetable juice, soy milk, fermented beverages), and natural seasonings(e.g., broth powder for ramen), but not limited thereto. Said food,beverages or food additives can be prepared in conventional manners.

The term “health functional food” is a group of food to which value isadded so as for the function thereof to be exerted and expressed for thepredetermined purpose by using physical, biochemical or bioengineeringtechniques thereto, or a processed food designed so as for the in-vivoadjustment functions of the relevant food composition such as rhythmadjustment in prophylaxis, prevention of disease and recovery fromdisease to be sufficiently expressed. Such functional food may comprisefood supplement additives which are food-scientifically acceptable, andmay additionally comprise suitable carriers, excipients and diluents,which are commonly used in the manufacturing thereof.

The term “functional beverages”, as used in the present invention,collectively refer to the drink products to relieve thirst or to enjoythe taste. There is no particular limitation thereto, except comprisingthe composition for improvement or prevention of said intestinal diseasesymptoms as essential ingredients with indicated ratio, and variousflavoring agents or natural carbohydrates may be contained therein asadditional ingredients like in common beverages.

In addition to the above, the food comprising the food compositionaccording to the present invention for improvement of said intestinaldisease symptoms or the prevention thereof may contain variousnutrients, vitamins, minerals (electrolyte), flavoring agents such assynthetic flavoring agents and natural flavoring agents, coloring agentsand fillers (cheese, chocolate, etc.), pectic acid and salts thereof,alginic acid and salts thereof, organic acids, protective colloidalthickening agents, pH adjusting agents, stabilizing agents,preservatives, glycerin, alcohol, carbonizing agents as used incarbonated beverages and the like, and each of the above ingredients maybe used alone or in combination with each other.

In the food comprising the food composition according to the presentinvention, the composition of the present invention may be comprised inan amount of 0.001% by weight to 100% by weight, and preferably 1% byweight to 99% by weight, based on the total weight of the food; in thecase of beverages, it may be comprised at an amount of 0.001 g to 10 g,and preferably 0.01 g to 1 g, based on 100 mL. For long-term intake forthe purpose of health and hygiene or for the purpose of health control,however, the amount may be below the above-mentioned range; and sincethe effective ingredients have no problem in terms of safety profile,they can be used at an amount above the range and they are not limitedto the amount range mentioned above.

The food composition according to the present invention may comprise theKBL382 strain, KBL384 strain, or KBL385 strain alone or in combinationwith the acceptable carrier, or may be prepared in the form of thecomposition suitable for consumption by human or animals. That is, thecomposition may be added to the food which contains no probioticbacteria or a couple of probiotic bacteria. For example, themicroorganisms which can be used in combination with the strainaccording to the present invention in preparing the food of the presentinvention should be suitable for the consumption by human or animals,and have probiotic activities to inhibit pathogenic, harmful bacteria orto improve the balance of microorganisms in the mammalian intestinaltract, upon intake, but not limited thereto. Such probioticmicroorganisms may include, for example, yeast such as Saccharomyces,Candida, Pichia and Torulopsis, fungi such as Aspergillus, Rhizopus,Mucor, and Penicillium, and bacteria belonging to the genus ofLactobacillus, Bifidobacterium, Leuconostoc, Lactococcus, Bacillus,Streptococcus, Propionibacterium, Enterococcus, and Pediococcus.Suitable probiotic microorganisms specifically may include, for example,Saccharomyces cerevisiae, Bacillus coagulans, Bacillus licheniformis,Bacillus subtilis, Bifidobacterium bifidum, Bifidobacterium infantis,Bifidobacterium longum, Enterococcus faecium, Enterococcus faecalis,Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacilluscasei, Lactobacillus curvatus, Lactobacillus delbruckii, Lactobacillusjohnsonii, Lactobacillus farciminus, Lactobacillus gasseri,Lactobacillus helveticus, Lactobacillus rhamnosus, Lactobacillusreuteri, Lactobacillus sakei, Lactococcus lactis, or Pediococcusacidilactici. Preferably, the food composition according to the presentinvention may further comprise a probiotic microorganism mixture havingexcellent probiotic activities and superior effects on enhancingimmunity to further enhance the effects thereof. The carriers that canbe included in the food composition of the present invention mayinclude, for example, extenders, high fiber additives, encapsulatingagents, and lipids, which are widely well known in the art. The strainof Lactobacillus paracasei in the present invention may be in thelyophilized or encapsulated form or in the form of culture suspensionsor dry powders.

The composition of the present invention can also be provided in theform of a feed additive comprising said strain or a feed comprising thesame.

The feed additive of the present invention may be in the form of dry orliquid formulation, and further comprise other non-pathogenicmicroorganisms in addition to the said strain of KBL382, KBL384, orKBL385. The microorganisms that can be added to the feed additive mayinclude, for example, Bacillus subtilis that can produce protease,lipase and sugar-converting enzymes, Lactobacillus strain having aphysiological activity and degradability of organic compounds underanaerobic conditions such as in the stomach of cow, filamentous fungisuch as Aspergillus oryzae showing effects on increasing weight oflivestock, milk yield, and digestibility of the feed (Slyter, L. L. J.Animal Sci. 1976, 43. 910-926) and yeast such as Saccharomycescerevisiae (Johnson, D. E et al. J. Anim. Sci., 1983, 56, 735-739;Williams, P. E. V. et al, 1990, 211).

The feed additive of the present invention may additionally comprise atleast one enzyme agent in addition to said Lactobacillus paracaseiKBL382 strain, Lactobacillus paracasei KBL384 strain, or Lactobacillusparacasei KBL385 strain. The additional enzyme agents can be in a dry orliquid form, and may include, for example, steatolytic enzymes such aslipase, phytase to produce phosphate and inositol phosphate by degradingphytic acid, amylase, i.e., an enzyme to hydrolyze a-1,4-glycoside bondincluded in, for example, starch and glycogen, phosphatase, i.e., anenzyme to hydrolyze organic phosphoric acid ester,carboxymethylcellulase to degrade cellulose, xylase to degrade xylose,maltase to hydrolyze maltose into two glucose molecules, and sugarproducing enzymes such as invertase to produce glucose-fructose mixtureby hydrolyzing saccharose.

In the use of the Lactobacillus paracasei KBL382 strain, Lactobacillusparacasei KBL384 strain, or Lactobacillus paracasei KBL385 strain of thepresent invention as feed additives, the raw ingredients for the feed,such as peanuts, peas, beets, pulp, grain by-products, animal gutspowder and fish meal powder, including various grains and soybeanprotein, can be used. They may be processed or not, and can be usedwithout limitation. The processing may include, but not limited thereto,such a process that the raw ingredients of the feed are charged and canbe compressed under pressure against a given outlet, and for proteins,extrusion by which proteins are degenerated to increase availability maybe preferably used. Extrusion has advantages of denaturing proteinsthrough thermal treatment and destroying antienzyme factors. Further,for soybean proteins, the digestibility thereof can be improved throughextrusion to inactivate anti-nutrients such as a trypsin inhibitor, oneof inhibitors of protease that are present in soybeans. Further,extrusion can promote improvement of digestibility by protease,enhancing the nutritional value of soybean proteins.

In another embodiment, the present invention relates to a pharmaceuticalcomposition for the treatment or prevention of intestinal diseases,comprising an effective amount of at least one selected from the groupconsisting of microbial cells of a Lactobacillus paracasei KBL382strain, a Lactobacillus paracasei KBL384 strain, or a Lactobacillusparacasei KBL385 strain, cultures of said strain, lysates of saidstrain, and extracts of said strain.

In another embodiment, the present invention relates to a pharmaceuticalcomposition for the treatment or prevention of allergic diseases,comprising an effective amount of at least one selected from the groupconsisting of microbial cells of a Lactobacillus paracasei KBL382strain, a Lactobacillus paracasei KBL384 strain, or a Lactobacillusparacasei KBL385 strain, cultures of said strain, lysates of saidstrain, and extracts of said strain.

In another embodiment, the present invention relates to a pharmaceuticalcomposition for the treatment or prevention of autoimmune diseases orinflammatory diseases, comprising an effective amount of at least oneselected from the group consisting of microbial cells of a Lactobacillusparacasei KBL382 strain, a Lactobacillus paracasei KBL384 strain, or aLactobacillus paracasei KBL385 strain, cultures of said strain, lysatesof said strain, and extracts of said strain.

The pharmaceutical composition of the present invention can be providedin a form of live bacteria, dry strain, cultures of said strain, lysatesof said strain, or a composition in combination with a pharmaceuticallyacceptable carrier or media. The carriers or media that can be usedherein may include solvent, a dispersant, a coating, an enhancer, acontrolled-release formulation (i.e., sustained-release formulation), orat least one inert excipient including starch, polyol, granules,microtine cellulose, microcrystalline cellulose such as Celphere,Celphere beads, diluent, lubricant, binder, disintegrant, and the like.The tablet of the above composition may be, if desired, coated by astandard aqueous or non-aqueous technique. The examples of thepharmaceutically acceptable carrier and the excipient for the use as thepharmaceutically acceptable inert carrier, and said additionalingredients may include, for example, a binder, a filler, adisintegrant, a lubricant, an antimicrobial agent and a coating agent,but not limited thereto.

The present invention may be characterized in that said intestinaldiseases are selected from the group consisting of abdominal bloating,abdominal discomfort, infectious diarrhea caused by pathogenicmicroorganisms, gastroenteritis, inflammatory bowel diseases,neurogenical intestinitis syndrome, irritable bowel syndrome, overgrowthof small intestinal microorganisms and intestinal feeding diarrhea, andthe diseases also include those caused by damage of barrier function ofintestine.

The inflammatory bowel disease (IBD) may include Crohn's disease, theintestinal lesion accompanying with Behcet's disease, ulcerativecolitis, hemorrhagic rectal ulcer, and pouchitis, and refers to a groupof diseases including Crohn's disease and ulcerative colitis. Ulcerativecolitis only affects the colon. Inflammation and ulcer of ulcerativecolitis are limited to the two innermost layers, mucosa and submucosaout of four layers of the colon. Inflammation and ulcer of Crohn'sdisease can be spread throughout all layers of the intestinal wall inboth small and large intestine.

Meanwhile, the irritable bowel syndrome is a chronic conditionaccompanying not only persistently recurrent abdominal discomfort andpain such as abdominal bloating, but also changes in bowel habit such asdiarrhea and constipation. The symptoms may be exacerbated bypsychological factors or stressful social circumstances.

In the present invention, the allergic diseases may include atopicdermatitis, urticaria, allergic rhinitis, allergic conjunctivitis,asthma, food allergy and anaphylactic shock, but not limited thereto.

In the present invention, autoimmune diseases may include, for example,rheumatoid arthritis, lupus, systemic scleroderma, atopic dermatitis,psoriasis, psoriatic arthritis, asthma, Guilian-Barre syndrome,myasthenia gravis, dermatomyositis, polymyositis, multiple sclerosis,autoimmune encephalomyelitis, polyarteritis nodosa, Hashimoto'sthyroiditis, multiple sclerosis, temporal arteritis, juvenile diabetes,alopecia areata, pemphigus, aphthous stomatitis, autoimmune hemolyticanemia, Wegener's granulomatosis, Sjogren's syndrome, Addison's disease,Crohn's disease, and Behcet's disease, but not limited thereto.

Further, inflammatory diseases of the present invention collectivelyrefer to conditions having inflammation as a main lesion, and mayinclude, for example, edema, conjunctivitis, periodontitis, rhinitis,otitis media, chronic sinusitis, pharyngolaryngitis, tonsillitis,bronchitis, pneumonia, gastric ulcer, gastritis, colitis, gout, eczema,acne, atopic dermatitis, contact dermatitis, seborrheic dermatitis,ankylosing spondylitis, rheumatic fever, fibromyalgia, osteoarthritis,psoriatic arthritis, rheumatoid arthritis, peri-arthritis of theshoulder, tendinitis, tenosynovitis myositis, hepatitis, cystitis,nephritis, Sjogren's syndrome, myasthenia gravis, sepsis, vasculitis,bursitis, temporal arteritis, and multiple sclerosis, but not limitedthereto.

It is known that autoimmune diseases can be prevented or treated by anactivation of regulatory T cells (Treg) or differentiation into T cells.Treg, a type of CD4+ T cell, plays a role in regulating the immunesystem and controlling autoimmune diseases by maintaining tolerance toautoantigens. A transcription factor called Foxp3 is specificallyinvolved in the development, maintenance, and function of regulatory Tcells, regulating the immune response through the secretion of theimmunosuppressive cytokines IL-10 and TGF-β.

In addition, it is known that when the differentiation rate of T cellsinto Th1 cells, Th2 cells, and Th17 cells is low and the differentiationrate of T cells into Treg cells is high, the inflammatory response canbe effectively suppressed.

Th1 cells, Th2 cells and Th17 cells differentiated from splenic T cellsare known to be associated with the expression of the followingtranscription factors and cytokines: In the case of Th1 cells, T-bet,IFN-γ and IL-12; in the case of Th2 cells, GATA3 and IL-5; in the caseof Th17 cells, RORγt, IL-17.

The present invention may be characterized in that the effects resultingfrom the strain of the present invention for alleviating, treating orpreventing autoimmune diseases or inflammatory diseases can be inducedby at least one selected from the following mechanisms:

i) suppression of expression or secretion of at least one selected fromthe group consisting of the inflammatory cytokines IL-6, TNF, IL-1b,IL-8, IL-2, IFN-γ, IL-4, IL-13, and IL-17A;

ii) increase of expression or secretion of the anti-inflammatory andimmunoregulatory cytokine IL-10; and

iii) increase of expression of Treg involved in anti-inflammatoryactivities and immunoregulation.

The term ‘treating’, unless mentioned otherwise, refers to reversing oralleviating the diseases or conditions used with said term or one ormore symptoms thereof, inhibiting the progression of the same orpreventing the same. The term ‘treatment’ as used in the presentinvention refers to an act of ‘treating’ as defined above. Accordingly,treatment or therapeutic regimen of intestinal diseases, allergicdiseases, autoimmune diseases or inflammatory diseases in mammals mayinclude one or more of the following:

(1) Inhibit the development of intestinal diseases, allergic diseases,autoimmune diseases or inflammatory diseases,

(2) Prevent the spread of the intestinal diseases, allergic diseases,autoimmune diseases or inflammatory diseases,

(3) Alleviate the intestinal diseases, allergic diseases, autoimmunediseases or inflammatory diseases,

(4) Prevent recurrence of the intestinal diseases, allergic diseases,autoimmune diseases or inflammatory diseases, and

(5) Palliate the symptoms of the intestinal diseases, allergic diseases,autoimmune diseases or inflammatory diseases

A composition of the present invention for preventing or treatingintestinal diseases, allergic diseases, autoimmune diseases orinflammatory diseases may comprise a pharmaceutically effective amountof a Lactobacillus paracasei KBL382 strain, a Lactobacillus paracaseiKBL384 strain, a Lactobacillus paracasei KBL385 strain alone or incombination with at least one of pharmaceutically acceptable carriers,excipients or diluents.

In the present invention, the term “effective amount” means an amountthat is high enough to provide a desired effect but is low enough toprevent serious side effects under medical judgment. The amount ofmicroorganisms administered to the body by the composition of thepresent invention can be appropriately adjusted in consideration of theadministration route and the administration target.

The composition of the present invention can be administered to asubject once or more per day. Unit dosage means physically discreteunits suitable for unit administration to human subjects and othermammals, and each unit comprises a suitable pharmaceutical carrier and apredetermined amount of the microorganisms of the present invention toprovide a therapeutic effect. The dosage unit for oral administration toan adult patient preferably contains 0.001 g or more of themicroorganism of the present invention, and the oral dosage of thecomposition of the present invention is from 0.001 g to 10 g, andpreferably from 0.01 g to 5 g per dose. The pharmaceutically effectiveamount of the microorganism of the present invention is from 0.01 g to10 g/day. However, the dosage varies depending on the severity of thepatient's disease and the microorganisms and auxiliary effectiveingredients used together. In addition, the total daily dosage can bedivided into several times and continuously administered as needed.Accordingly, the above dosage ranges do not limit the scope of thepresent invention in any way.

Further, the term “pharmaceutically acceptable” as used above refers toa composition that is physiologically acceptable and does not cause anallergic reaction such as gastrointestinal disorder, dizziness, orsimilar reaction when administered to a human.

A composition of the present invention can be formulated using methodsknown in the art so that rapid, sustained or delayed release of theactive ingredients, after administered to a mammal, can be provided. Thedosage forms may be powders, granules, tablets, emulsions, syrups,aerosols, soft or hard gelatin capsules, sterile injection solutions, orsterile powders. Further, the composition of the present invention forpreventing or treating intestinal diseases can be administered viaseveral routes, including oral, transdermal, subcutaneous, intravenousor intramuscular administration. The dosage of the active ingredientscan be appropriately selected depending on various factors such as theroute of administration, the patient's age, sex, weight, and theseverity of the patient. The composition of the present invention forpreventing or treating gastrointestinal diseases can be administered incombination with a known compound having the effect of preventing ortreating the symptoms of intestinal diseases.

The pharmaceutical composition of the present invention, in particular,can be provided in an oral unit dosage form of an enteric coatedformulation. The term “enteric coating”, as used herein, comprises anyknown pharmaceutically acceptable coating which can remain in thestomach without degrading by the gastric acid and can sufficientlydisintegrate in the intestinal tract to release active ingredientstherein. The “enteric coating” of the present invention refers to acoating that can be maintained for 2 hours or more when an artificialgastric juice such as an HCl solution of pH 1 is contacted thereto at36° C. to 38° C., and subsequently can degrade, preferably in anartificial intestinal juice such as a KH₂PO₄ buffer solution of pH 6.8in 30 minutes.

The enteric coating of the present invention is coated on one core in anamount of from about 16 mg to 30 mg, preferably from 16 mg to 20 mg or25 mg or less. When the thickness of the enteric coating of the presentinvention is 5 μm to 100 μm, and preferably 20 μm to 80 μm, satisfactoryresults can be obtained as an enteric coating. The material of theenteric coating can be suitably selected from known polymeric materials.Suitable polymeric materials are listed in a number of known documents(L. Lachman et al., The Theory and Practice of Industrial Pharmacy,3^(rd) ed., 1986, pp. 365-373; H. Sucker et al., PharmazeutischeTechnologie, Thieme, 1991, pp. 355-359; Hagers Handbuchderpharmazeutischen Praxis, 4^(th) ed., Vol. 7, pp. 739-742, and 766-778,(SpringerVerlag, 1971); and Remington's Pharmaceutical Sciences, 13^(th)ed., pp. 1689-1691 (Mack Publ., Co., 1970)), and cellulose esterderivatives, cellulose ethers, a copolymer of acrylic resin andmethylacrylate, and a copolymer of maleic acid and phthalic acidderivatives can be included therein.

The enteric coating of the present invention can be prepared using aconventional enteric coating method in which an enteric coating solutionis sprayed onto a core. Suitable solvents used for the enteric coatingprocess are alcohols such as ethanol, ketones such as acetone,halogenated hydrocarbon solvents such as dichloromethane (CH₂Cl₂), andmixed solvents of these solvents. A softener such as di-n-butylphthalate or triacetin is added to the coating solution in a ratio of1:about 0.05 to about 0.3 (coating material:softener). It is appropriateto carry out the spraying process continuously, and it is possible toadjust the spraying amount in consideration of the conditions ofcoating. The spraying pressure can be variously adjusted, andsatisfactory results can be obtained generally with a spraying pressureof about 1 bar to about 1.5 bar.

In another embodiment, the present invention relates to the use of saidstrain or composition for the prevention or treatment of intestinaldiseases, allergic diseases, autoimmune diseases or inflammatorydiseases, and the use of said strain or composition for preparing atherapeutic agent for intestinal diseases, allergic diseases, autoimmunediseases or inflammatory diseases.

Specifically, the present invention relates to a composition for the useof preventing or treating intestinal diseases, comprising at least oneselected from the group consisting of a Lactobacillus paracasei KBL382strain (Accession No. KCTC13509BP), a Lactobacillus paracasei KBL384strain (Accession No. KCTC13510BP), or a Lactobacillus paracasei KBL385strain (Accession No. KCTC13511BP), cultures of said strain, lysates ofsaid strain, and extracts of said strain.

In addition, the present invention relates to a composition for the useof preventing or treating allergic diseases, comprising at least oneselected from the group consisting of a Lactobacillus paracasei KBL382strain (Accession No. KCTC13509BP), a Lactobacillus paracasei KBL384strain (Accession No. KCTC13510BP), or a Lactobacillus paracasei KBL385strain (Accession No. KCTC13511BP), cultures of said strain, lysates ofsaid strain, and extracts of said strain.

In addition, the present invention relates to a composition for the useof preventing or treating autoimmune diseases or inflammatory diseases,comprising at least one selected from the group consisting of aLactobacillus paracasei KBL382 strain (Accession No. KCTC13509BP), aLactobacillus paracasei KBL384 strain (Accession No. KCTC13510BP), or aLactobacillus paracasei KBL385 strain (Accession No. KCTC13511BP),cultures of said strain, lysates of said strain, and extracts of saidstrain.

The present invention also relates to the use of a compositioncomprising at least one selected from the group consisting of aLactobacillus paracasei KBL382 strain (Accession No. KCTC13509BP), aLactobacillus paracasei KBL384 strain (Accession No. KCTC13510BP), or aLactobacillus paracasei KBL385 strain (Accession No. KCTC13511BP),cultures of said strain, lysates of said strain, and extracts of saidstrain, for preparing a preventive or therapeutic drug for intestinaldiseases.

In addition, the present invention relates to the use of a compositioncomprising at least one selected from the group consisting of aLactobacillus paracasei KBL382 strain (Accession No. KCTC13509BP), aLactobacillus paracasei KBL384 strain (Accession No. KCTC13510BP), or aLactobacillus paracasei KBL385 strain (Accession No. KCTC13511BP),cultures of said strain, lysates of said strain, and extracts of saidstrain, for preparing a preventive or therapeutic drug for allergicdiseases.

In addition, the present invention relates to the use of a compositioncomprising at least one selected from the group consisting of aLactobacillus paracasei KBL382 strain (Accession No. KCTC13509BP), aLactobacillus paracasei KBL384 strain (Accession No. KCTC13510BP), or aLactobacillus paracasei KBL385 strain (Accession No. KCTC13511BP),cultures of said strain, lysates of said strain, and extracts of saidstrain, for preparing a preventive or therapeutic drug for autoimmunediseases or inflammatory diseases.

The term ‘prevention’, as used herein, is associated with averting,delaying, impeding or hindering diseases to reduce the same.

The term ‘treatment’, as used herein, is associated with caring for asubject suffering from diseases in order to ameliorate, cure or reducethe symptoms of the diseases or to reduce or stop the progression of thediseases.

In another embodiment, the present invention provides a method forpreventing or treating intestinal diseases, allergic diseases,autoimmune diseases or inflammatory diseases, comprising administering apharmaceutically effective amount of said strain or said composition toa subject in need of prevention or treatment of said diseases, or inneed of alleviation of the intestinal health, allergic reactions,decreased level of immunity or inflammatory reactions.

Specifically, the present invention provides a method for treatingintestinal diseases, comprising administering a therapeuticallyeffective amount of at least one selected from the group consisting of aLactobacillus paracasei KBL382 strain (Accession No. KCTC13509BP), aLactobacillus paracasei KBL384 strain (Accession No. KCTC13510BP), or aLactobacillus paracasei KBL385 strain (Accession No. KCTC13511BP),cultures of said strain, lysates of said strain, and extracts of saidstrain to a subject in need thereof.

In addition, the present invention provides a method for treatingallergic diseases, comprising administering a therapeutically effectiveamount of at least one selected from the group consisting of aLactobacillus paracasei KBL382 strain (Accession No. KCTC13509BP), aLactobacillus paracasei KBL384 strain (Accession No. KCTC13510BP), or aLactobacillus paracasei KBL385 strain (Accession No. KCTC13511BP),cultures of said strain, lysates of said strain, and extracts of saidstrain to a subject in need thereof.

In addition, the present invention provides a method for treatingautoimmune diseases or inflammatory diseases, comprising administering atherapeutically effective amount of at least one selected from the groupconsisting of a Lactobacillus paracasei KBL382 strain (Accession No.KCTC13509BP), a Lactobacillus paracasei KBL384 strain (Accession No.KCTC13510BP), or a Lactobacillus paracasei KBL385 strain (Accession No.KCTC13511BP), cultures of said strain, lysates of said strain, andextracts of said strain to a subject in need thereof.

Since the pharmaceutical composition used for the method for preventingor treating said intestinal diseases, allergic diseases, autoimmunediseases or inflammatory diseases, and the administration method thereofhave been described above, the overlapping contents between thecomposition and the method will be omitted herein to avoid excessivecomplexity of the present specification.

Meanwhile, the said subject to which the composition for preventing ortreating said intestinal diseases, allergic diseases, autoimmunediseases or inflammatory diseases can be administered includes allanimals including human. For example, the subject may be an animal suchas dog, cat, or mouse.

Hereinafter, the present invention will be described in more detailthrough examples. These examples are only for illustrating the presentinvention, and it will be apparent to those skilled in the art that thescope of the present invention is not to be construed as being limitedby these examples.

Example 1. Screening of Probiotic Strains with Immunoregulatory Function

Probiotic strains having an immunoregulatory function were screenedusing the leukemia monocyte THP-1 cell line and peripheral bloodmononuclear cells (PBMC). Strains derived from human gut or vagina,respectively, were seeded to the above two cell lines so that the ratioof cell line: strain was 1:100, and the ratio of IL-10, a major cytokineindicating inflammation control, to IL-6, a major cytokine indicating aninflammatory reaction (IL-10/IL-6), and the ratio of IL-10 to IFN-γ, amajor cytokine indicating autoimmune reaction (IL-10/IFN-γ) weremeasured. A total of 23 strains were used for the screening as follows:two Lactobacillus gasseri strains, one Lactobacillus reuteri strain,five Lactobacillus rhamnosus strains, two Lactobacillus fermentumstrains, four Lactobacillus paracasei strains, four Lactobacillussalivarius strains, one Lactobacilus plantarum strain, two Lactobacillusacidophilus strains and two Lactococcus lactis strains.

As a result of the screening, the highest IL-10/IL-6 values were shownin the three strains of Lactobacillus paracasei KBL382, KBL384 andKBL385. In particular, among the three Lactobacillus paracasei strains,KBL382 had the highest IL-10/IL-6 value of 2.22 in the THP-1 cell line,and the IL-10/IFN-γ value was 9 in peripheral blood mononuclear cells,showing a very high immunoregulatory effect. Table 1 below shows theresults of the IL-10/IL-6 value and IL-10/IFN-γ value measured for threeLactobacillus paracasei strains out of the twenty three probioticstrains used in the present screening. Accordingly, three strains ofLactobacillus paracasei KBL382, KBL384 and KBL385 were predicted to havea high effect on improving intestinal health, and additional experimentswere conducted on these strains.

TABLE 1 Cell line Strain IL-10/IL-6 THP-1 Lactobacillus paracasei KBL3822.222168 Lactobacillus paracasei KBL384 0.621436 Lactobacillus paracaseiKBL385 1.08215 Cell Strain IL-10/IFN-r PBMC Lactobacillus paracaseiKBL382 9.0295403 Lactobacillus paracasei KBL384 7.4016942 Lactobacillusparacasei KBL385 7.9818579

Example 2. Verification of the Effect of Lactobacillus paracasei KBL382,KBL384 and KBL385 Strains on Inflammatory Cytokine Expression and T-CellDifferentiation Regulation (In Vitro Test)

In order to verify the immunoregulatory effect from said Lactobacillusparacasei KBL382, KBL384 and KBL385 strains, the ability to generatecytokines involved in immunoregulation was confirmed in THP-1 cell linetreated with the Lactobacillus paracasei KBL382, KBL384 and KBL385strains, while whether the marker gene of T-cell differentiation wasexpressed was confirmed in PBMC treated with the Lactobacillus paracaseiKBL382 and KBL385 strains.

First, the THP-1 cell line was seeded onto each well of a 24-well plateby 1×10⁵ cells, differentiated into mature macrophages, and then theculture solution was replaced. After 3 hours, one well was treated withLPS at a concentration of 1 μg/mL as a positive control, and the otherwells were treated with the Lactobacillus paracasei KBL382, KBL384 orKBL385 strain, each by 1×10⁷ cells based on the number of viablebacteria; after 24 hours, the culture solution was collected and theamount of each cytokine was measured using the BD Cytometric Bead Array(CBA) human inflammation kit (Cat No. 551811) according to themanufacturer's method. As a negative control (Cont), the well wastreated with PBS buffer and the same experimental procedure wasperformed. As a result, as shown in FIGS. 1A-1E, the measurement showedthat a group of THP-1 cell lines treated with Lactobacillus paracaseiKBL382 and KBL385 strains had a significantly lower amount ofinflammatory cytokines IL-6, TNF, IL-1b and IL-8 than the LPS-treatedgroup. In the case of the treatment with the KBL384 strain, it wasconfirmed that IL-6 was significantly lower than the test group treatedwith LPS, but TNF, IL-1b and IL-8 were not different significantly fromthe group treated with LPS. In the case of IL-10, any significantincrease or decrease was not observed in the groups treated with theKBL382, KBL384 and KBL385 as compared to the group treated with LPS, butit was observed that overall increase in IL-10 level was induced ascompared to the negative control group. Therefore, it was concluded thatLactobacillus paracasei KBL382 and KBL385 strains were effective forsignificantly suppressing the generation of inflammatory cytokines inTHP-1 cells, and the subsequent experiment was conducted to verify thepattern of T cell differentiation.

In order to confirm the effect of the KBL382 and KBL385 strains on Tcell differentiation, PBMC cells were seeded onto each well of a 24-wellplate by 5×10⁵ cells, and then the Lactobacillus paracasei KBL382 orKBL385 strain was added by 5×10⁶ cells. As a control, E. coli (E. coli0157 EC4115) was added to each well by 5×10⁶ cells based on the numberof viable cells, or LPS was added at a concentration of 500 ng/mL. E.coli is known to increase the expression of T-bet, GATA3, and RORγtgenes, which are effector cell markers of T cells related toinflammatory response, while LPS is known to increase the expression ofFOXP3 gene, a marker of Treg cells related to inflammation regulation.The PBMC cell test group prepared under the above conditions wasincubated for 5 days and then the cells were obtained and collected. Inorder to confirm the level of gene expression, first, the Easy-spin™(DNA free) Total RNA Extraction Kit (Intron) was used to extract RNA andthen the High Capacity RNA-to-cDNA Kit (ThermoFisher) was used tosynthesize cDNA. A real-time PCR was conducted on the synthesized cDNAwith the Rotor-Gene SYBR Green PCR kit (Qiagen) using a Rotor-Gene® Q(Qiagen) equipment according to the manufacturer's method and the mRNAexpression of T-bet which is Th1 marker (effector cell marker) gene,GATA3 which is Th2 marker gene, RORγt which is Th17 marker gene, andFOXP3 which is Treg cell marker gene were measured. At this time, theexpression level of B2M genes was measured as an internal control tocorrect the relative gene expression level between each test group. Thebase sequence of the primer used to confirm the expression of each geneis as follows.

B2M Forward: (SEQ ID NO. 4) 5′-CCA GCA GAG AAT GGA AAG TC-3′′ Reverse:(SEQ ID NO. 5) 5′-GAT GCT TCT TAC ATG TCT CG-3′ T-bet Forward:(SEQ ID NO. 6) 5′-CCC CAA GGA ATT GAC AGT TG-3′ Reverse) (SEQ ID NO. 7)5′-GGG AAA CTA AAG CTC ACA AAC-3′ GATA3 Forward: (SEQ ID NO. 8)5′-CTG CAA TGC CTG TGG GCT C-3′ Reverse: (SEQ ID NO. 9)5′-GAC TGC AGG GAC TCT CGC T-3′ RORγt Forward: (SEQ ID NO. 10)5′-AAG ACT CAT CGC CAA AGC AT-3′ Reverse: (SEQ ID NO. 11)5′-TCC ACA TGC TGG CTA CAC A-3′ FOXP3 Forward: (SEQ ID NO. 12)5′-TCA AGC ACT GCC AGG CG-3′ Reverse: (SEQ ID NO. 13)5′-CAG GAG CCC TTG TCG GAT-3′

As a result, as shown in FIGS. 2A-2D, it was confirmed that theLactobacillus paracasei KBL382 and KBL385 strains, compared to E. coli,maintained the expression level of T-bet, GATA3, and RORγt genes, whichare effector cell markers of inflammatory T cells, at a significantlylow level, but significantly increased mRNA of FOXP3, a marker of Tregcells for maintaining immune homeostasis similarly to LPS.

Example 3. Verification of the Effect of Lactobacillus paracasei KBL382,KBL384 and KBL385 Strains on T-Cell Immunoregulation (In Vitro Test)

Meanwhile, in order to verify T-cell immunoregulatory effect byLactobacillus paracasei KBL382, KBL384 and KBL385 strains, theexpression level of the inflammatory cytokines was verified when T-cellswere activated in PBMC cells, and then followed by the treatment withsaid paracasei KBL382, KBL384 and KBL385 strains.

PBMC cells were seeded onto each well of a 96-well plate by 2×10⁵ cellswith an anti-CD3 antibody (1 μg/mL, ebioscience) to activate T cells,and then a Lactobacillus paracasei KBL382, KBL384 or KBL385 strain wasadded by 1×10⁷ cells to be “bacteria:PBMC=50:1”, based on the number ofviable cells. As a control, Escherichia coli K12 (ATCC 10798) was addedto each well by 1×10⁷ cells based on the number of viable cells. Afterincubating the test group of T cells-activated PBMC cells prepared underthe above conditions for 3 days, the supernatant was collected and theamount of each cytokine was measured using the BD cytometric Bead Array(CBA) human inflammation kit (Cat No. 551811) according to themanufacturer's method.

As a result, as shown in FIGS. 3A-3E, in the PBMC cell lines thatT-cells were activated by addition of an anti-CD3 antibody, theLactobacillus paracasei KBL382, KBL384 and KBL385 strains allsignificantly decreased the expression of IL-2, which is a cytokine ofTh1, IL-4 and IL-13, which are respectively cytokines of IFNγ and Th2,and IL-17A, which is a cytokine of Th17, as compared to the test grouptreated only with the E. coli-added strain and PBS, while theanti-inflammatory cytokine IL-10 was remarkably increased in all of thethree Lactobacillus paracasei strains used in the experiment, ascompared to the test group treated only with PBS; it was confirmed thatespecially the KBL 382 strain, among the three, significantly increasedIL-10 as compared to the E. coli added group. The experiment results asabove led to a conclusion that all of the Lactobacillus paracaseiKBL382, KBL384 and KBL385 strains significantly decreased inflammatorycytokines of T-cells and increased anti-inflammatory cytokines.

Example 4. Verification of the Effect of Lactobacillus paracasei KBL382,KBL384 and KBL385 Strains on Enteritis Alleviation Effects (In VivoTest)

The present example was to confirm whether the Lactobacillus paracaseiKBL382, KBL384 and KBL385 strains showed the intestinal functionimprovement effects even in vivo. To this end, C57BL/6 mice were dividedinto groups of 10 mice each, and then fed tap water with 2% DSSdissolved therein for 9 days, and thereby inducing enteritis. At thesame time, the mice in the control group were orally administered with200 μL of PBS daily, and the mice in the test group were daily providedvia an oral administration with 200 μL of each of the Lactobacillusparacasei KBL382, KBL384 or KBL385 strain which was diluted in PBS to be2×10¹⁰ CFU/mL so that the amount of daily administration could be set at4×10⁹ CFU. Then, during the 9 days in which enteritis was induced byDSS, the body weight changes of the mice in the control group and testgroup were measured daily, and on the 9^(th) day after DSS was supplied,mice were subjected to autopsies to measure the length of the colon.

As a result, as shown in FIGS. 4A-4E, it was confirmed that regardingthe body weight changes, the groups treated with KBL382, KBL384 andKBL385 strains all showed a significant effect on alleviating the weightloss, as compared to the mice in the control group with no treatment;regarding the colon length change, the width of decrease in the colonlength was significantly improved in all the groups of three strains, ascompared to the mice in the control group.

In addition, H&E staining was performed to measure the degree ofinflammation in the test groups of KBL382 and KBL385 strains, andMyeloperoxidase (MPO) levels were measured in the tissues. Afterautopsy, in order to examine the lesions of the colon by H&E staining,the distal part of the colon was fixed in 10% neutral formalin solution,and then paraffin tissue specimens were sectioned with a thickness of 5μm, stained with hematoxylin & eosin, and observed with an opticalmicroscope. Further, for MPO measurement, colon tissue was, first, putin RIPA buffer to which a protease inhibitor was added, and wasdisrupted with a homogenizer. After centrifuging the disrupted tissue at4° C. at 15,000×g for 10 minutes, MPO was measured using the supernatantaccording to the protocol of ELISA kit (Hycult Biotech, MPO, Mouse, cat.HK210-02).

As a result, as shown in FIGS. 4A-4E, it was confirmed through the H&Estaining results, that the test groups that the two strains, KBL382 andKBL385, were administered all alleviated inflammation as compared to thecontrol group, and the level of MPO was slightly decreased in the KBL385test group and significantly decreased in the KBL382 test group. Usingthe colon tissues thus obtained were used to confirm the strengtheningeffect on tight junction of the intestinal tract wall.

Example 5. Effects of the Lactobacillus paracasei KBL382 and KBL385Strains on Strengthening Intestinal Tract Wall Tight Junction

In order to verify the effects of the Lactobacillus paracasei KBL382 andKBL385 strains on strengthening intestinal tract wall tight junction,the mRNA expression levels of genes involved in the intestinal tractwall tight junction were compared and measured in the cells isolatedfrom the mouse colon tissue isolated in Example 4. To check the amountof gene expression, RNA was first extracted from tissues using theEasy-spin™ (DNA free) Total RNA Extraction Kit (Intron), and then cDNAwas synthesized with the High Capacity RNA-to-cDNA Kit (ThermoFisher).The Rotor-gene SYBR green PCR kit (Qiagen) was used to measure in vivothe mRNA expression level of zonula occluden-1 (ZO-1), Claudin3 andMUC-4, which are the intestinal tract wall tight junction markerproteins, using a Rotor-Gene® Q (Qiagen). The expression level of thehypoxanthine-guanine phosphoribosyl-transferase (HPRT) gene was measuredas a control to normalize the relative gene expression level betweeneach test groups. The primer used to confirm the expression is preparedto be able to specifically bind to each gene as follows.

Zo-1 Fw: (SEQ ID No. 14) 5′-ACC CGA AAC TGA TGC TGT GGA TAG-3′  Rw:(SEQ ID No. 15) 5′-AAA TGG CCG GGC AGA ACT TGT GTA-3′ Claudin3 Fw:(SEQ ID No. 16) 5′-CAG ACG TCC GTC AGT TTT CG-3′ Rw: (SEQ ID No. 17)5′-CAT GGC TGC TGG ACT TGA AC-3′ MUC-4 Fw: (SEQ ID No. 18)5′-GTC TCC CAT CAC GGT TCA GT-3′ Rw: (SEQ ID No. 19)5′-TGT CAT TCC ACA CTC CCA GA-3′ HPRT Fw: (SEQ ID No. 20)5′-TTA TGG ACA GGA CTG AAA GAC-3″ Rw: (SEQ ID No. 21)5′-GCT TTA ATG TAA TCC AGC AGG T-3″

As a result, as shown in FIGS. 5A-5C, it was found that the levels ofthe three genes measured in the mice dosed with the KBL382 and KBL385strains were increased as compared to the control group dosed with onlyPBS, and thereby the intestinal tract wall tight junction was recovered;accordingly, said strains can be used for treating the diseases such asIBD and IBS.

Example 6. Comparison of the Enteritis Alleviating Effects Between anTherapeutic Antibody for Enteritis and KBL382

Infliximab (product name Remicade) is a therapeutic recombinant antibodydrug used as an injection for autoimmune diseases such as rheumatoidarthritis, ankylosing spondylitis, ulcerative colitis, Crohn's diseasein adults, Crohn's disease in children, psoriasis, and psoriaticarthritis. The purpose of this study was to confirm the difference ineffect on alleviating intestinal disease symptoms in vivo between theKBL 382 strain and the infliximab.

For the comparison of the effects, after dividing C57BL/6 mice intogroups of eight mice, the mice were fed tap water with 2% DSS dissolvedtherein for 9 days, inducing enteritis. At the same time, 200 μL of PBSwas orally administered to the mice in the control group daily, while200 μL of the KBL382 strain diluted in PBS to be 2×10¹⁰ CFU/mL wasorally administered to the mice in the test group daily so that theamount of daily administration could be set at 4×10⁹ CFU. To thetherapeutic antibody administration group, infliximab antibody wasadministered once on Day 3 to be a dose of 5 mg/kg per mouse.

Later, during the 9 days in which enteritis was induced by DSS, the bodyweight changes of the mice in the control group and test group weremeasured daily, and on the 9^(th) day after DSS was supplied, mice weresubjected to autopsies to measure the length of the colon.

As a result, as shown in FIGS. 6A and 6B, regarding the colon lengthchange, it was confirmed that the width of decrease in the colon lengthwas significantly improved in the groups that KBL382 strain was treatedand that infliximab was administered, compared to the mice in thecontrol group. It was confirmed that the effect of improving the colonlength of the group that KBL382 strain was treated was similar or ratherhigher than the group that infliximab was administered.

Regarding the change in body weights, as shown in FIG. 6C, the effect onbody weight decrease was significantly improved in the groups thatKBL382 strain was treated and that infliximab was administered, comparedto the mice in the control group with no treatment. It was confirmedthat the effect of alleviating the initial weight loss during five daysafter the administration was superior in the group that infliximab wasadministered over the group that KBL382 strain was treated, but laterthe improvement effect by infliximab was decreased, while the effect ofthe KBL382 strain-treated group on the weight loss was increased,showing similar effects between the two groups until the 9^(th) day.Accordingly, regarding the use of said strain for the treatment ofinflammatory intestine diseases and irritable bowel syndrome, it wasconfirmed that a therapeutic effect similar to the commerciallyavailable therapeutic antibodies could be expected.

Example 7. Effects of KBL382 on Alleviation of Atopic Conditions

In order to verify the effect on atopic alleviation of the KBL382strain, the NC/Nga mouse model, an animal model of atopic skin disease,was used.

After dividing NC/Nga mice into groups of nine mice, the back of eachmouse was epilated from the lower ear to the upper tail and mice wereleft for 24 hours. Then, an ointment comprising Dermatophagoides farinaeextract (DFE), a house dust mite (HDM) antigen, was applied once ortwice a week for seven weeks onto the epilated portion so that eachmouse could be treated with 100 mg of DFE, and thereby inducing atopicdermatitis. From the third week of dermatitis induction, 200 μL of PBSwas orally administered to the mice in the control group daily; each ofthe KBL382 strain and Lactobacillus rhamnosus KBL365 strain, which wasdiluted in 200 μL of PBS to be at least 1×10⁹ CFU/mouse, was orallyadministered to the mice in the test group by 200 μL daily. Then, duringfour weeks of administration of the bacteria, dermatitis scores of themice in the control and test group were measured weekly, and on the4^(th) week after the administration of the bacteria, the mouse'sscratching time and skin thickness, and IgE concentration-in-blood afterconducting autopsies of mice were measured.

7-1. Evaluation of Dermatitis Score

To evaluate DFE-induced skin lesions, the dermatitis score was measuredthrough the following method. Skin conditions were monitored by takingpictures for 4 weeks at one week intervals from the 3rd week since thestrain had been administered. Four indicators of dryness, edema,erythema/hemorrhage, and erosion/excoriation of the skin were checked.The total score was evaluated with 0 points for no lesion, 1 point formild, 2 points for moderate, and 3 points for severe.

As a result, as shown in FIGS. 7A and 7B, the dermatitis score wassignificantly reduced in the group dosed with KBL382 strain, compared tothe control group (negative) where atopic dermatitis was induced, andthe group that the KBL365 strain was administered. As a result, theeffect of alleviating atopic dermatitis score according to the intake ofKBL382 strain was verified.

7-2 Effects on Alleviating Itching Symptoms

In order to verify the effect of alleviating itching according to theadministration of KBL382 strain in the mouse models suffering fromatopic dermatitis induced by DFE, the number of scratches was measuredfor the mouse during 10 minutes after 4 weeks of strain administration.

As a result, as can be seen in FIG. 7C, it appeared that the number ofscratches was significantly reduced in the test group that KBL382 wasadministered, as compared to the PBS administration group, whichconfirmed that the itching symptoms of atopic dermatitis were alleviatedby the administration of the KBL382 strain.

7-3. Decrease of Skin Thickness

In order to verify the effect of decreasing the skin thickness afteradministration of KBL382 to mouse models suffering from atopicdermatitis induced by DFE, the mouse ear thickness and dorsal skinthickness were measured with calipers four weeks after the strain wasadministered, and the relief of edema symptom due to atopic dermatitiswas observed. As a result, as can be seen in FIGS. 7D and 7E, it wasobserved that in the test group dosed with KBL382, the ear and skinthicknesses were significantly reduced, as compared to the control groupand the KBL365-administered group.

7-4. Decrease in IgE Concentration-in-Blood

It has been found that the concentration of IgE in patients havingatopic dermatitis has mostly increased as clinical severity of atopicdermatitis increased (Matsumoto M, J. Immunol. 1999). Thus, theconcentration of IgE, a representative hematologic factor appearing asatopic dermatitis arises, was measured by collecting blood three weeksafter the strain was administered, separating serum therefrom and usingMouse IgE ELISA Set (Cat No. 555248, BD OptEIA™). As a result, as shownin FIG. 7F, it was found that the concentration of IgE-in-blood wassignificantly decreased in the test group that KBL382 was orallyadministered, which indicated the effect of treating atopic dermatitisafter intake of KBL382.

Specific aspects of the present invention have been described in detailabove, and it is obvious to those skilled in the art that these specificaspects are only preferred embodiments, and the scope of the presentinvention is not limited thereby. Therefore, the scope of the presentinvention is substantially defined by the following claims, withequivalents to the claims.

Name of Depository Organization: Korean Collection for Type Cultures,Korea Research Institute of Bioscience and Biotechnology (KRIBB), 181,Ipsin-gil, Jeongeup-si, Jeolllabuk-do, 56212, Republic of Korea

Accession No.: KCTC13509BP

Accession Date: 20180417

Name of Depository Organization: Korean Collection for Type Cultures,Korea Research Institute of Bioscience and Biotechnology (KRIBB), 181,Ipsin-gil, Jeongeup-si, Jeolllabuk-do, 56212, Republic of Korea

Accession No.: KCTC13510BP

Accession Date: 20180417

Name of Depository Organization: Korean Collection for Type Cultures,Korea Research Institute of Bioscience and Biotechnology (KRIBB), 181,Ipsin-gil, Jeongeup-si, Jeolllabuk-do, 56212, Republic of Korea

Accession No.: KCTC13511BP

Accession Date: 20180417

INDUSTRIAL APPLICABILITY

The strains of Lactobacillus paracasei KBL382 (Accession No.KCTC13509BP), Lactobacillus paracasei KBL384 (Accession No.KCTC13510BP), and Lactobacillus paracasei KBL385 (Accession No.KCTC13511BP) according to the present invention have excellentanti-inflammatory and immunomodulatory functions, has a superbstrengthening effect on tight junction of the intestinal tract wall,suppresses enteritis-induced weight loss and colon length reduction,thereby exhibiting a therapeutic effect for enteritis, and weakensallergic reaction of celss, and significantly alleviates the symptoms ofatopic dermatitis, and thereby providing improvement of allergicdiseases. Therefore, a single strain can achieve enhancinganti-inflammatory effects, strengthening immunity, improving intestinalhealth functions, and alleviating allergic diseases, and thus can beuseful as a probiotic material.

Sequence List Free Text

An electronic file of Sequence List attached

The invention claimed is:
 1. An enteric coated dosage form comprising anenteric coated core, the core comprising an immunoregulatory amount ofat least one of Lactobacillus paracasei KBL382 with Accession No.KCTC13509BP or cultures of said strain.
 2. The enteric coated dosageform according to claim 1, wherein the immunoregulatory amount of theenteric dosage form is sufficient for treating or preventing intestinaldisease, allergic disease, autoimmune disease or inflammatory disease.3. The enteric coated dosage form according to claim 2, wherein saidintestinal disease is selected from abdominal bloating, abdominaldiscomfort, infectious diarrhea caused by pathogenic microorganisms,gastroenteritis, inflammatory bowel diseases, neurogenical intestinitissyndrome, irritable bowel syndrome, overgrowth of small intestinalmicroorganisms or intestinal feeding diarrhea.
 4. The enteric coateddosage form according to claim 2, wherein said allergic disease isatopic dermatitis, urticaria, allergic rhinitis, allergicconjunctivitis, asthma, food allergy or anaphylactic shock.
 5. A methodfor treating an intestinal disease, comprising administering atherapeutically effective amount of the enteric coated dosage formaccording to claim 1 or cultures of said strain to a subject in needthereof.
 6. A method for treating an allergic disease, comprisingadministering a therapeutically effective amount of the enteric coateddosage form according to claim 1 or cultures of said strain to a subjectin need thereof.
 7. A method for treating an autoimmune disease or aninflammatory disease, comprising administering a therapeuticallyeffective amount of the enteric coated dosage form according to claim 1or cultures of said strain to a subject in need thereof.